The state of contractile activation or relaxation of cardiac and skeletal muscle is controlled by rise or reduction of the Ca2+ concentration in the myoplasm. The aim of this program is to characterize the mechanism by which Ca2+ is removed from the myoplasm to produce relaxation, and alternatively released into the myoplasm to produce contractile activation. One section of the program deals with sarcoplasmic reticulum, which is the membrane system involved in Ca2+ removal by active transport. The catalytic and transport cycle of the Ca2+ dependent ATPase of sarcoplasmic reticulum will be studied in detail, the partial reactions characterized by equilibrium and kinetic methods, and the catalytic mechanism investigated by chemical modification of reactive enzyme residues. Methods of immunology and molecular genetics will be also used to generate monoclonal antibodies, clone and analyze the cDNAs and the genes encoding the ATPase, study the expression of the ATPase following gene transfer in mammalian systems, and obtain alterations of the DNAs encoding the ATPase to generate material for structure-function and bioregulation studies. Another section of the program deals with the mechanism of Ca2+ release from sarcoplasmic reticulum. Fiber preparations and microsomal fractions derived from various sections of the sarcotubular membrane system will be tested for their ability to release Ca2+ as required for contractile activation. The nature of the signal triggering release and the characteristics of the channels involved in release will be investigated. The kinetics of release will be studied by measurements of radioactive tracer fluxes, and recording electrical currents in reconstituted membrane bilayers and patch clamp experiments. Isolation of channel protein will be attempted.